SummaryCoumarins are derived via the phenylpropanoid pathway in plants. The 2H-1-benzopyran-2-one core structure of coumarins is formed via the ortho-hydroxylation of cinnamates, trans/cis isomerization of the side chain, and lactonization. Ortho-hydroxylation is a key step in coumarin biosynthesis as a branch point from lignin biosynthesis; however, ortho-hydroxylation of cinnamates is not yet fully understood. In this study, scopoletin biosynthesis was explored using Arabidopsis thaliana, which accumulates scopoletin and its b-glucopyranoside scopolin in its roots. T-DNA insertion mutants of caffeoyl CoA O-methyltransferase 1 (CCoAOMT1) showed significant reduction in scopoletin and scopolin levels in the roots, and recombinant CCoAOMT1 exhibited 3¢-O-methyltransferase activity on caffeoyl CoA to feruloyl CoA. These results suggest that feruloyl CoA is a key precursor in scopoletin biosynthesis. Ortho-hydroxylases of cinnamates were explored in the oxygenase families in A. thaliana, and one of the candidate genes in the Fe(II)-and 2-oxoglutaratedependent dioxygenase (2OGD) family was designated as F6¢H1. T-DNA insertion mutants of F6¢H1 showed severe reductions in scopoletin and scopolin levels in the roots. The pattern of F6¢H1 expression is consistent with the patterns of scopoletin and scopolin accumulation. The recombinant F6¢H1 protein exhibited orthohydroxylase activity for feruloyl CoA (K m = 36.0 AE 4.27 lM; k cat = 11.0 AE 0.45 sec )1 ) to form 6¢-hydroxyferuloylCoA, but did not hydroxylate ferulic acid. These results indicate that Fe(II)-and 2-oxoglutarate-dependent dioxygenase is the pivotal enzyme in the ortho-hydroxylation of feruloyl CoA in scopoletin biosynthesis.

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Clinical evidence suggests that cortical excitability is increased in depressives. We investigated its cellular basis in a mouse model of depression. In a modified version of forced swimming (FS), mice were initially forced to swim for 5 consecutive days and then were treated daily with repetitive transcranial magnetic stimulation (rTMS) or sham treatment for the following 4 weeks without swimming. On day 2 through day 5, the mice manifested depression-like behaviors. The next and last FS was performed 4 weeks later, which revealed a 4 week maintenance of depression-like behavior in the sham mice. In slices from the sham controls, excitability in cingulate cortex pyramidal cells was elevated in terms of membrane potential and frequencies of spikes evoked by current injection. Depolarized resting potential was shown to depend on suppression of large conductance calcium-activated potassium (BK) channels. This BK channel suppression was confirmed by measuring spike width, which depends on BK channels. Chronic rTMS treatment during the 4 week period significantly reduced the depression-like behavior. In slices obtained from the rTMS mice, normal excitability and BK channel activity were recovered. Expression of a scaffold protein Homer1a was reduced by the FS and reversed by rTMS in the cingulate cortex. Similar recovery in the same behavioral, electrophysiological, and biochemical features was observed after chronic imipramine treatment. The present study demonstrated that manifestation and disappearance of depression-like behavior are in parallel with increase and decrease in cortical neuronal excitability in mice and suggested that regulation of BK channels by Homer1a is involved in this parallelism.